The proposed studies are designed to study a novel cysteine protease, expressed by certain Streptococcus pyogenes isolates. This enzyme, designated IdeS (immunoglobulin degrading enzyme of streptococcus), has the ability to bind to IgG, IgA and IgM but only selectively cleave antibodies of the IgG isotype. The selective substrate specificity is further exemplified by its reported inability to cleave a synthetic peptide consisting of the precise amino acid sequence surrounding the known clip site in IgG. Recent studies suggest that some of these unusual properties may be due to the need to form a complex between two IdeS molecules and a single substrate IgG molecule for catalysis to occur. [unreadable] [unreadable] The focus of the proposed research is to characterize the functional properties of IdeS more fully. The initial focus (specific aim #1) will be to use a rapid, sensitive, semi-quantitative SELDI-TOF assay developed in our laboratory to measure the activity of a wild type and recombinant form of IdeS, isolated or cloned from a M1 serotype S. pyogenes isolate, AP1. This assay will facilitate detailed analysis of the optimal ratio of reactants for generation of Fc fragments. [unreadable] [unreadable] The second specific aim will focus on characterizing isolates that secrete IdeS and the effects of IdeS on free IgG versus IgG contained within an antigen-antibody complex. These studies are designed to help elucidate the potential roles of IdeS in the pathogenesis of S. pyogenes and the potential impact of a secreted IgG-degrading enzyme on the host-pathogen interaction. [unreadable] [unreadable] The third specific aim seeks to use bioinformatics data mining approaches, cloning and expression of related genes from other species of streptococci that infect horses and other animals to determine if this potential pathogenic factor is conserved among related streptococcal species that target different hosts. The focus of these studies will be to determine if the related gene products have IgG-degrading potential and, if so, demonstrate any difference in substrate preference towards immunoglobulin from the hosts they preferential infect. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: This is valuable project for introducing students to cross-disciplinary approaches to a research problem in biology. The studies involve a combination of techniques in molecular biology, proteomics, enzymology and bioinformatics. The studies have relevance to basic immunochemistry, as well as having potential implications for host-pathogen interactions of an important human pathogen [unreadable] [unreadable] [unreadable] [unreadable]